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. 1982;1(9):1023–1028. doi: 10.1002/j.1460-2075.1982.tb01290.x

Subclasses of simian virus 40 large T antigen: differential binding of two subclasses of T antigen from productively infected cells to viral and cellular DNA.

E Fanning, K H Westphal, D Brauer, D Cörlin
PMCID: PMC553156  PMID: 6329720

Abstract

Two major subclasses of simian virus 40 (SV40) large T antigen were separated by zone velocity sedimentation of crude extracts from productively infected cells. These subclasses, which have been shown to differ biologically and biochemically ( Fanning et al., 1981), sedimented at 5-6S and 14-16S. The amount of T antigen in each form was estimated by complement fixation and by immunoprecipitation of T antigen from extracts of cells chronically labeled with [35S]methionine. Each form of T antigen was tested for specific binding to end-labeled restriction fragments of SV40 DNA using an immunoprecipitation assay. The 5-6S and 14-16S forms of T antigen both bound specifically to DNA sequences in the SV40 HindIII C fragment. The sequences required for binding both forms were localized in the same 35-bp region of the origin. However, significant differences in binding activity and affinity for specific and nonspecific DNA were demonstrated. These properties suggest that T antigen subclasses may serve different functions in the lytically infected cell.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradley M. K., Griffin J. D., Livingston D. M. Relationship of oligomerization to enzymatic and DNA-binding properties of the SV40 large T antigen. Cell. 1982 Jan;28(1):125–134. doi: 10.1016/0092-8674(82)90382-8. [DOI] [PubMed] [Google Scholar]
  2. Chaconas G., Van de Sande J. H., Church R. B. End group labelling of RNA and double stranded DNA by phosphate exchange catalyzed by bacteriophage T4 induced polynucleotide kinase. Biochem Biophys Res Commun. 1975 Oct 6;66(3):962–969. doi: 10.1016/0006-291x(75)90734-2. [DOI] [PubMed] [Google Scholar]
  3. Cole C. N., Landers T., Goff S. P., Manteuil-Brutlag S., Berg P. Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro. J Virol. 1977 Oct;24(1):277–294. doi: 10.1128/jvi.24.1.277-294.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DiMaio D., Nathans D. Cold-sensitive regulatory mutants of simian virus 40. J Mol Biol. 1980 Jun 15;140(1):129–142. doi: 10.1016/0022-2836(80)90359-9. [DOI] [PubMed] [Google Scholar]
  5. Fanning E., Nowak B., Burger C. Detection and characterization of multiple forms of simian virus 40 large T antigen. J Virol. 1981 Jan;37(1):92–102. doi: 10.1128/jvi.37.1.92-102.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gidoni D., Scheller A., Barnet B., Hantzopoulos P., Oren M., Prives C. Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA. J Virol. 1982 May;42(2):456–466. doi: 10.1128/jvi.42.2.456-466.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Greenspan D. S., Carroll R. B. Complex of simian virus 40 large tumor antigen and 48,000-dalton host tumor antigen. Proc Natl Acad Sci U S A. 1981 Jan;78(1):105–109. doi: 10.1073/pnas.78.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hansen U., Tenen D. G., Livingston D. M., Sharp P. A. T antigen repression of SV40 early transcription from two promoters. Cell. 1981 Dec;27(3 Pt 2):603–613. doi: 10.1016/0092-8674(81)90402-5. [DOI] [PubMed] [Google Scholar]
  9. Harlow E., Pim D. C., Crawford L. V. Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells. J Virol. 1981 Feb;37(2):564–573. doi: 10.1128/jvi.37.2.564-573.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  11. Hobom G. Replication signals in prokaryotic DNA. Curr Top Microbiol Immunol. 1981;94-95:93–142. doi: 10.1007/978-3-642-68120-2_3. [DOI] [PubMed] [Google Scholar]
  12. Kuchino T., Yamaguchi N. Characterization of T antigen in cells infected with a temperature-sensitive mutant of simian virus 40. J Virol. 1975 Jun;15(6):1302–1307. doi: 10.1128/jvi.15.6.1302-1307.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  15. McCormick F., Harlow E. Association of a murine 53,000-dalton phosphoprotein with simian virus 40 large-T antigen in transformed cells. J Virol. 1980 Apr;34(1):213–224. doi: 10.1128/jvi.34.1.213-224.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McKay R. D. Binding of a simian virus 40 T antigen-related protein to DNA. J Mol Biol. 1981 Jan 25;145(3):471–488. doi: 10.1016/0022-2836(81)90540-4. [DOI] [PubMed] [Google Scholar]
  17. McKay R., DiMaio D. Binding of an SV40 T antigen-related protein to the DNA of SV40 regulatory mutants. Nature. 1981 Feb 26;289(5800):810–813. doi: 10.1038/289810a0. [DOI] [PubMed] [Google Scholar]
  18. Montenarh M., Henning R. Simian virus 40 T-antigen phosphorylation is variable. FEBS Lett. 1980 May 19;114(1):107–110. doi: 10.1016/0014-5793(80)80870-2. [DOI] [PubMed] [Google Scholar]
  19. Myers R. M., Kligman M., Tjian R. Does simian virus 40 T antigen unwind DNA? J Biol Chem. 1981 Oct 10;256(19):10156–10160. [PubMed] [Google Scholar]
  20. Myers R. M., Rio D. C., Robbins A. K., Tjian R. SV40 gene expression is modulated by the cooperative binding of T antigen to DNA. Cell. 1981 Aug;25(2):373–384. doi: 10.1016/0092-8674(81)90056-8. [DOI] [PubMed] [Google Scholar]
  21. Myers R. M., Tjian R. Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6491–6495. doi: 10.1073/pnas.77.11.6491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Myers R. M., Williams R. C., Tjian R. Oligomeric structure of a simian virus 40 T antigen in free form and bound to DNA. J Mol Biol. 1981 Jun 5;148(4):347–353. doi: 10.1016/0022-2836(81)90180-7. [DOI] [PubMed] [Google Scholar]
  23. Oren M., Winocour E., Prives C. Differential affinities of simian virus 40 large tumor antigen for DNA. Proc Natl Acad Sci U S A. 1980 Jan;77(1):220–224. doi: 10.1073/pnas.77.1.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Osborn M., Weber K. SV40: T antigen, the A function and transformation. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):267–276. doi: 10.1101/sqb.1974.039.01.035. [DOI] [PubMed] [Google Scholar]
  25. Prives C., Beck Y., Gidoni D., Oren M., Shure H. DNA binding and sedimentation properties of SV40 T antigens synthesized in vivo and in vitro. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):123–130. doi: 10.1101/sqb.1980.044.01.014. [DOI] [PubMed] [Google Scholar]
  26. Reich N. C., Levine A. J. Specific interaction of the SV40 T antigen-cellular p53 protein complex with SV40 DNA. Virology. 1982 Feb;117(1):286–290. doi: 10.1016/0042-6822(82)90531-1. [DOI] [PubMed] [Google Scholar]
  27. SEVER J. L. Application of a microtechnique to viral serological investigations. J Immunol. 1962 Mar;88:320–329. [PubMed] [Google Scholar]
  28. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  29. Shalloway D., Kleinberger T., Livingston D. M. Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion. Cell. 1980 Jun;20(2):411–422. doi: 10.1016/0092-8674(80)90627-3. [DOI] [PubMed] [Google Scholar]
  30. Shenk T. E., Carbon J., Berg P. Construction and analysis of viable deletion mutants of simian virus 40. J Virol. 1976 May;18(2):664–671. doi: 10.1128/jvi.18.2.664-671.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shenk T. A biochemical method for increasing the size of deletion mutations in simian virus 40 DNA. J Mol Biol. 1977 Jul 5;113(3):503–515. doi: 10.1016/0022-2836(77)90235-2. [DOI] [PubMed] [Google Scholar]
  32. Shortle D. R., Margolskee R. F., Nathans D. Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6128–6131. doi: 10.1073/pnas.76.12.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tegtmeyer P., Andersen B., Shaw S. B., Wilson V. G. Alternative interactions of the SV40 A protein with DNA. Virology. 1981 Nov;115(1):75–87. doi: 10.1016/0042-6822(81)90090-8. [DOI] [PubMed] [Google Scholar]
  34. Tjian R. Protein-DNA interactions at the origin of simian virus 40 DNA replication. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):655–661. doi: 10.1101/sqb.1979.043.01.073. [DOI] [PubMed] [Google Scholar]
  35. Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978 Jan;13(1):165–179. doi: 10.1016/0092-8674(78)90147-2. [DOI] [PubMed] [Google Scholar]
  36. Twigg A. J., Sherratt D. Trans-complementable copy-number mutants of plasmid ColE1. Nature. 1980 Jan 10;283(5743):216–218. doi: 10.1038/283216a0. [DOI] [PubMed] [Google Scholar]
  37. Wensink P. C., Finnegan D. J., Donelson J. E., Hogness D. S. A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell. 1974 Dec;3(4):315–325. doi: 10.1016/0092-8674(74)90045-2. [DOI] [PubMed] [Google Scholar]
  38. Winter R. B., Berg O. G., von Hippel P. H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 3. The Escherichia coli lac repressor--operator interaction: kinetic measurements and conclusions. Biochemistry. 1981 Nov 24;20(24):6961–6977. doi: 10.1021/bi00527a030. [DOI] [PubMed] [Google Scholar]
  39. Yang Y. C., Hearing P., Rundell K. Cellular proteins associated with simian virus 40 early gene products in newly infected cells. J Virol. 1979 Oct;32(1):147–154. doi: 10.1128/jvi.32.1.147-154.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]

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